Hemolysis scavenger proteins and renal function marker in children with sickle cell disease at steady state: A cross‐sectional study

Abstract Background and Aims Hemolysis is a fundamental feature of sickle cell disease (SCD) contributing to the vaso‐occlusive crisis of patients. The objectives of the study were to assess the link between hemolysis proteins and hematological parameters, and to validate cystatin C (CYS C) as a potent renal marker in diagnoising SCD. Method Here, a cross‐sectional study carried out at the pediatric SCD clinic of the Komfo Anokye Teaching Hospital comprised 90 SCD children (HbSC, HbSF, and HbSS). ANOVA, t‐test, and Spearman's rank correlation analysis were done. Elevated proteins levels were compared to standard values; alpha‐1 microglobulin (A1M) (1.8−65 µg/L), CYS C (0.1−4.5 µmol/L), and haemopexin (HPX) (500−1500 µg/mL). Results The mean (standard deviation) age of participants was 9.830 (±0.3217) years, and 46% of them were males. From simple descriptive analysis, we observed that all but one patient had their HPX level below the reference range (<500 µg/mL). Here, A1M levels were shown to be within the appropriate reference range for all the patients except few patients. CYS C levels were also all within the required reference values. A Spearman's rank correlation test between full blood count and HPX generally suggested a weak but positive correlation; RBC (coef. = 0.2448; p = 0.0248), HGB (coef. = 0.2310; p = 0.030), hematocrit (coef. = 0.2509; p = 0.020), and platelet (coef. = 0.1545; p = 0.160). Mean corpuscular volume (coef. = ‐0.5645; p = 0.610) had a stronger but negative correlation with HPX. This study depicts a positive and stronger association between CYS C and HPX levels (coef. = 0.9996; p < 0.0001), validating the use of CYS C as a useful marker of renal function in persons with SCDs. Conclusion In the present study, we show that A1M levels were normal for most of the patients, hence CYS C levels are not alarming in this study. Further, there exists a correlation between hemolysis scavenger proteins and hematological parameters.


| INTRODUCTION
Sickle cell disease (SCD) is a genetic form of anemia in which mutated forms of hemoglobin distort the red blood cells into crescent shapes at low oxygen levels. About 300,000 babies are born each year with sickle cell anemia, with 75% of this population in Africa. 1 In every 5 people with SCD, 1 develops renal abnormalities which could lead to chronic kidney disease (CKD). 2 There are several variants of SCD: Sickle cell anemia (HbSS), sickle hemoglobin-C (HbSC), and sickle faetal hemoglobin (HbSF).
The HbSC form of SCD is mostly common in Africa. 3 In general, patients with the HbSC form of the disease have milder abnormalities than their homozygous (HbSS) counterparts due to less anemia. 4,5 Delays in diagnosis of SCD may increase the risk for developing CKD eventually leading to increased morbidity and mortality. 6 Although renal complications in sickle cell remains painless and develops gradually over time, its effect results in organ dysfunction.
As a vaso-occlusive disease, the contribution of anemia and hemolysis to the progression of both acute and chronic sickle cell complications cannot be underestimated. [7][8][9] Anemia is a common presentation in SCD; resulting mainly from hemolysis, and is a reflection of disease severity. 9,10 Intravascular hemolysis in SCD is marked by the scavenging activities of danger associated molecular patterns (DAMPs) (i.e., haptoglobin [HPG], haemopexin [HPX], and alpha-1 microglobulin [A1M]). 7,9 HPG and HPX neutralize the DAMPs through the liver, with A1M stepping in only when HPX becomes depleted. Hemolytic anemia in SCD may also increase with malaria infection. 11 Despite the detrimental contribution of anemia and hemolysis to organ damage, there is no hematological technology to determine marked injury. The aforementioned scenarios suggest that levels of the hemolysis scavenger proteins (HSPs) could be the only indication of intravascular hemolysis. 12 Hemolysis due to reduced/depleted HPX, and high levels of A1M has been linked to poor kidney function in sickle mice studies, and further stated that, this incident coupling with anemia may also lead to kidney dysfunction and damage. 8,12 The serum levels of urea and creatinine are commonly used as renal function tests. This notwithstanding, studies have shown that serum levels of cystatin C (CYS C) are a more precise test of renal function (as represented by the glomerular filtration rate, GFR) than urea and creatinine. [13][14][15] A study on a cohort of pediatric SCD patients in Ghana rather used urea and creatinine to assess kidney function. 12 Thus, previous study conducted on organ damage in SCD (ORDISS), may not be able to detect mild renal impairment, and predict risk of developing CKD, as the serum levels of urea and creatinine are inaccurate at detecting this complication. Thus, there is the need to study the effect of SCD on renal function using a biomolecule whose serum levels provides a precise understanding on the function of the kidneys, especially, at the early stages of renal complication. Furthermore, studies that have reported on anemia and hemolysis in relation to kidney function and other organ functions in SCD have mostly involved mouse models and/or SCD patients 8,16,17 outside of Ghana; hence, there is minimal data, if any, on Ghanaian SCD patients. This warrants the need to provide such laboratory data on HSPs in relation to kidney function in this cohort. Provision of such data and knowledge from this study will contribute to improving the management of SCD patients, and as well serve as a pedestal for further studies on the laboratory presentations of pediatric SCD patients at steady state. Here, we showed that hemolysis is mainly followed by depletion in HPX levels and a rise in A1M levels.
Furthermore, we also observed a link between HPX and CYS levels validating CYS C as a useful renal marker.

| Study design
This was a cross-sectional study carried out from July 2021 to November 2021 at the pediatric SCD clinic at the Komfo Anokye Teaching Hospital (KATH). The study comprised 90 children with SCD. After explaining the study procedures and obtaining informed consent from participants, screening questionnaires and case report forms were administered to collect data (age, sex, sickle status, malaria, FBC, and HSPs measurement).
The population were children with confirmed SCDs attending clinic visits at KATH. Children with SCD of all sexes, in a steady-state and from the ages of 5−14 years were recruited and included in the study. Steady-state was defined as the absence of clinical symptoms or steady-state clinically confirmed by a physician.

| Sample size estimation and sampling technique
The minimum sample size estimated for the study was 90. This was determined using the Fisher's formula for sample size calculation, N = [z 2 p (1−p)]/d, 218 using a reported prevalence of 6% 6 of steady-state children with SCD. Specifically, N was the minimum sample size estimated; z was the point of standard normal distribution curve which was set at 1.96 (95% confidence interval); p was the assumed prevalence rate; and d was the degree of precision which was set at 6%. This study employed a random sampling technique in recruiting study participants. The registry of the sickle cell clinic was reviewed for selection of all participants.

| Data collection
Caregivers and/or study participants were interviewed with an electronic semistructured questionnaire hosted on the School of Medicine and Dentistry, KNUST, Research Electronic Data Capture (REDCap) server. 19 REDCap is a secure web application for building and managing online surveys and databases. To ensure that quality data was collected, the questionnaires were mainly answered by caregivers on behalf of study participants below the ages of 13 years whilst participants from 13 years and above responded to questionnaire and in some cases were assisted by their caregivers. Moreover, questions were interpreted in the local language where necessary for easy comprehension. Medical records were reviewed for each study participant for the clinic visit to confirm their steady state and complete other relevant variables. The questionnaire was categorized into demographic data, clinical characteristics, and medical history. Furthermore, the laboratory results of study participants were entered into a laboratory documentation sheet hosted on REDCap.

| Demographics of study participants
A total of 90 participants with SCD were recruited into this study (HbSS, n = 56), (HbSC, n = 25), and (HbSF, n = 9). The age of participants ranged from 5 to 14 years with a mean age of 9.830 (±3.018) years. There was a significance difference in ages (p < 0.001).
Moreover, among the patients, HbSC (p < 0.010) and HbSS (p < 0.010) were significantly older in years than HbSF patients. We observed that 80% of the patients were on hydroxyurea treatment, with a higher proportion being HbSS variant as shown in Table 1.
The full blood count parameters were compared with their respective reference values (Table 2).

| Description of hematological parameters among SCD patients in steady state
To assess the occurrence of anemia among the SCD variants, we compared RBC, HGB, hematocrit (HCT), mean corpuscular volume (MCV), platelet (PLT), and white blood count (WBC) values to their respective reference range (   (Table 3).
To manage SCD properly, hemolysis must be closely monitored.  (Table 4).

| Low mean corpuscular volume predicts iron deficiency among SCD at steady state
High mean corpuscular volume is an indicator of iron stores in SCD patients, especially after hydroxyurea treatment, however, we observed low MCV in a higher proportion of our patients. In  Figure 2B.

| Interdependence of HSP and renal function marker in SCD
This part establishes an association between HPX and CYS, both of which are known to measure specific liver and kidney activities, respectively. From Figure 3A, there was a positive and stronger association between CYS C and HPX levels (coef. = 0.9996; p < 0.001). This shows that CYS C level is dependent on HPX levels. Figure 3B also shows a stronger and positive correlation between A1M levels and HPX levels (coef. = 0.9995; p < 0.001).  circulate in the blood long enough as normal ones do, leading to lower hemoglobin levels. 24 A recent report from our laboratory shows that successive blood transfusion during anemia is usually accompanied by iron overload in SCD. 25 In SCD, there is an increased tendency for RBC lysis and adhesion, thereby leading to hemolysis. A Positive association was observed between HPX and A1M as shown in Table 3, however, this association was weak. The extent of hemolysis is driven by RBC instability and the extent of potential heme toxicity by changes in heme that affect it's binding to globin. 26 Chronic hemolysis usually seen in SCD shorten red cell survival as well as low erythropoietin in SCD, reducing levels of HGB and HCT as observed in our present study. 21 The findings in this study are in accordance with other studies in Africa and other low-income and middle-income countries that showed significant impairment in RBC and other hematological indices. 26 Indeed, we have shown that total red RBC count, hemoglobin (HGB) concentration, HCT count, and MCV were substantially reduced in almost 65% of patients from this study. 26,27 Interestingly, low levels of MCV as seen here in a higher proportion of patients could reflect low compliance of hydroxyurea treatment in patients from this study as some reported of breakages in treatment.

| DISCUSSION
Free heme drives endothelial cell expression of adhesion molecules to which platelets and RBCs attach, ultimately blocking blood flow. 28 Here, we observed a total decline in HPX levels in almost all SCD patients except one. The downregulation of HPX (the scavenger protein of cell free haeme) is an indication of hemolysis in SCD. There were no statistical differences among the SCD cohort on gender and age comparison, nonetheless higher levels of HPX were seen in the females and the older age group. This supports findings that there is no statistic differences in HPX levels between males and females. 28,29 This scenario buttresses our observation of HPX depletion in almost all SCD cohort in this study, confirming HPX levels an indicator of hemolysis in hemolytic diseases such as SCD.
Studies have also shown that adult levels of HPX are attained 6 months after birth, 17 and thus, confirms that adult levels of HPX were already attained in all study participants preceding our study (age criteria ranged from 4 to 14 years).
The main role of A1M is to take over the detoxification activity of free haeme through the kidney when HPX is depleted. 8,28 Therefore, low levels of HPX but high A1M levels denote hemolysis. From this study, we observed that as HPX levels get depleted, A1M levels were gradually rising, however, levels were considerably within the appropriate reference range. 28 Mouse model study has reported that excess heme is usually directed to the liver in healthy mice while excess heme travels to the kidney in SCD mice leading to chronic kidney damage. 30 In the present study, our data indicate that as HPX levels get depleted, A1M levels rises gradually, denoting a positive and stronger correlation between HPX and A1M. Elsewhere, it has been reported that A1M levels are increased 1.6-folds in SCD as compared to healthy controls. 31 The commonest renal diagnostic approach for estimating GFR is creatinine. Due to its lack of sensitivity for early detection of CKD, it has become important to find other markers that are potent in diagnosing kidney dysfunction earlier especially in SCD. [34][35][36] Studies utilizing CYS C as a renal functional marker in SCD are few, however, reports indicates they are sensitive than other renal markers. 13,37 A study using 20 cohort of SCD children established a correlation between CYS C and albuminuria, 38 and later found CYS C to be more sensitive. 13 Here, we showed that most of our patients' CYS C levels were within reference range. This observation could be as a result of (A) (B)

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data and materials are available in the corresponding author's institution and will be made available upon formal request.

ETHICS STATEMENT
Ethical clearance was obtained from the KATH Institutional Review Board (IRB) with reference: KATH IRB/CA/064/21. The background, aims, and study procedures were thoroughly explained to caregivers and patients.
Consent and assent were obtained before any study participant was enrolled onto the study. Any study-related procedure/documentation was completed in compliance with the appropriate regulatory authorities.
Recruitment and sampling procedures was conducted in accordance with the WHO guidelines for good laboratory practice.

TRANSPARENCY STATEMENT
The lead author Fatima A. Fordjour affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.